Intracellular delivery system for protein phosphatases

Abstract

The subject invention relates to a polymer-based intracellular delivery system for protein phosphatases. This delivery system can be used to deliver protein phosphatases for any disease or disorder in which it is desirable to elevate protein phosphatase activity, particularly for tumor therapy. Novel protein phosphatases are also disclosed.

What is claimed is:  
     
         1 . A complex molecule comprising a conjugate of a polymer capable of being taken up by a cell linked to a protein phosphatase polypeptide, the conjugate capable of achieving intracellular delivery of the polypeptide while retaining its biological activity.  
     
     
         2 . The conjugate of  claim 1 , wherein the polymer is linked to the polypeptide by means of a direct chemical bond or a linker.  
     
     
         3 . The conjugate of  claim 2 , wherein the linker is not degraded under physiological conditions.  
     
     
         4 . The conjugate of  claim 2 , wherein the linker comprises a peptide.  
     
     
         5 . The conjugate of  claim 4 , wherein the peptide is Gly-Gly.  
     
     
         6 . The conjugate of  claim 1 , wherein the polymer is an N-alkyl acrylamide polymer.  
     
     
         7 . The conjugate of  claim 6 , wherein the N-alkyl acrylamide polymer is a homopolymer.  
     
     
         8 . The conjugate of  claim 6 , wherein the N-alkyl acrylamide polymer is a copolymer.  
     
     
         9 . The conjugate of  claim 8 , wherein the copolymer comprises N-hydroxypropyl methacrylamide.  
     
     
         10 . The conjugate of  claim 1 , wherein the protein phosphatase enzyme is PP2C.  
     
     
         11 . The conjugate of  claim 10 , wherein the protein phosphatase is selected from the group consisting of PP2Cα, PP2Cβ, PP2Cγ (also called FIN 13), PP2Cδ, Wip1, Ca ++ -calmodulin dependent kinase II phosphatase, NER PP-2C, and PP2Cζ-zeta.  
     
     
         12 . A pharmaceutical composition comprising as an active ingredient a complex molecule comprising a conjugate of a polymer capable of being taken up by a cell linked to a protein phosphatase polypeptide, the conjugate capable of achieving intracellular delivery of the polypeptide while retaining its biological activity, together with a pharmaceutically acceptable carrier or diluent.  
     
     
         13 . The pharmaceutical composition of  claim 12 , wherein the polymer is linked to the polypeptide by means of a direct chemical bond or a linker.  
     
     
         14 . The pharmaceutical composition of  claim 13 , wherein the linker is not degraded under physiological conditions.  
     
     
         15 . The pharmaceutical composition of  claim 13 , wherein the linker comprises a peptide.  
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein the peptide is Gly-Gly.  
     
     
         17 . The pharmaceutical composition of  claim 12 , wherein the polymer is an N-alkyl acrylamide polymer.  
     
     
         18 . The pharmaceutical composition of  claim 17 , wherein the N-alkyl acrylamide polymer is a homopolymer.  
     
     
         19 . The pharmaceutical composition of  claim 17 , wherein the N-alkyl acrylamide polymer is a copolymer.  
     
     
         20 . The pharmaceutical composition of  claim 19 , wherein the copolymer comprises N-hydroxypropyl methacrylamide.  
     
     
         21 . The pharmaceutical composition of  claim 12 , wherein the protein phosphatase enzyme is PP2C.  
     
     
         22 . The pharmaceutical composition of  claim 21 , wherein the protein phosphatase is selected from the group consisting of PP2Cα, PP2Cβ, PP2Cγ (also called FIN 13), PP2Cδ, Wip1, Ca ++ -calmodulin dependent kinase II phosphatase, NER PP-2C, and PP2Cζ-zeta.  
     
     
         23 . Use for the preparation of a medicament of a complex molecule comprising a conjugate of a polymer capable of being taken up by a cell linked to a protein phosphatase polypeptide, the conjugate capable of achieving intracellular delivery of the polypeptide while retaining its biological activity.  
     
     
         24 . Use according to  claim 23  of a complex molecule comprising a conjugate according to any one of claims  2 - 11 .  
     
     
         25 . A method of treating a subject suffering from a disease or disorder which comprises administering to the subject a therapeutically effective amount of a composition comprising as an active ingredient a complex molecule comprising a conjugate of a polymer capable of being taken up by a cell linked to a protein phosphatase polypeptide, the conjugate capable of achieving intracellular delivery of the polypeptide while retaining its biological activity.  
     
     
         26 . The method of  claim 25  wherein the disease is a tumor.  
     
     
         27 . The method of  claim 25  wherein the composition is according to any one of claims  13 - 22 .  
     
     
         28 . The method of  claim 26  wherein the composition is according to any one of claims  13 - 22 .  
     
     
         29 . The complex molecule of  claim 1  further comprising a protein localization signal.  
     
     
         30 . The complex molecule of  claim 29  wherein the protein localization signal is an internal protein localization signal.  
     
     
         31 . The complex molecule of  claim 1  further comprising at least one anti-cancer agent or targeting agent.  
     
     
         32 . The composition of  claim 12  further comprising at least one additional anti-cancer agent.  
     
     
         33 . A polypeptide comprising the amino acid sequence of SEQ ID NO 3 or SEQ ID NO 5.  
     
     
         34 . A biologically active composition comprising the polypeptide of  claim 34 .  
     
     
         35 . A DNA fragment which encodes the polypeptide of  claim 33 .  
     
     
         36 . The DNA fragment of  claim 35  comprising the nucleotide sequence of SEQ ID NO 2 or SEQ ID NO 4.  
     
     
         37 . An expression vector comprising the DNA fragment of  claim 35 , and suitable regulatory elements positioned so as to effect expression of the polypeptide encoded by said DNA fragment.  
     
     
         38 . An expression vector comprising the DNA fragment of  claim 36 , and suitable regulatory elements positioned so as to effect expression of the polypeptide encoded by said DNA fragment.  
     
     
         39 . A DNA fragment which hybridizes with the DNA fragment of  claim 35 .  
     
     
         40 . A DNA fragment which hybridizes with the DNA fragment of  claim 36.